In the kidney, the polarity of the tubular epithelial cells is essential for the absorption and secretion of ions and solute. The asymmetric organization of the plasma membrane into apical and basolateral domains possessing different compositions of membrane proteins, transporters, and lipids makes vectorial flow across the epithelium possible. When cell polarity is compromised by nephrotoxic or ischemic injury, or by other disease processes, the ability of the nephron to function is lost. The cellular and molecular events which permit the biogenesis of epithelial cell polarity are not completely understood. It is likely, however, that the microtubule cytoskeleton is intimately involved because it is known that microtubules play a role in polarized intracellular transport and because the microtubule cytoskeleton dramatically rearranges during cell polarization from a juxtanuclear architecture to one arranged along the apical-basal axis. The long term objectives of this project are to investigate what part microtubules play in the biogenesis of epithelial cell polarity. In this application the role of microtubule associated proteins (MAPs) in the organization and function of the microtubule cytoskeleton will be examined. In Specific Aim 1, the function of MAP-4 (also called MAP-U), which has been shown to be expressed in the kidney, will be investigated by overexpression of native, mutated, and antisense MAP-4 constructs, and by blocking of MAP-4 activity in permeabilized MDCK cells using specific antibodies. Efforts will also be made to identify, purify, and prepare reagents against other epithelial cell MAPs. In Specific Aim 2, the three-dimensional organization of the distinctive apical cap and vertical polarized microtubules in MDCK cells will be visualized using high-resolution electron microscopy of rapid-frozen, deep- etched specimens. Interactions of microtubules with the actin cytoskeleton and specific cytoskeletal-binding proteins in the apical and basal cell cortices will be looked at by gold immunolabeling of freeze-etched cells. In Specific Aim 3, the role of the microtubule motors kinesin and cytoplasmic dynein in polarized exocytosis will be studied by addition of activity-blocking antibodies to permeabilized MDCK cells still able to transport proteins from the Golgi complex to the plasma membrane.